The present invention relates to a method and apparatus for producing lipid vesicles. More particularly, the invention is directed to a production technique that permits rapid, high volume formation of lipid vesicles from surfactants and other amphiphilic molecules.
Lipid vesicles are lipid structures which surround and encapsulate aqueous volumes. There are many uses for these structures, e.g., as adjuvants or as carriers for the transportation of encapsulated drugs or biologically-active substances. Lipid vesicles are often classified into three groups by size and structure: multilamellar vesicles ("MLV's"), large unilamellar vesicles ("LUV's"), and small unilamellar vesicles ("SUV's"). MLV's are onion-like structures having a series of substantially spherical shells formed of lipid bilayers interspersed with aqueous layers. LUV's have a diameter greater than 1.mu. and are formed of a lipid bilayer surrounding a large, unstructured aqueous phase. SUV's are similar in structure to the LUV's except their diameters are less than 0.2.mu..
A fourth type of lipid vesicle, which is particularly well suited for transport of either lipids or aqueous materials, is the paucilamellar vesicle ("PLV"). See, Callow and McGrath, Cryobiology 1985, 22(3), pp. 251-267. This type of vesicle has an external structure of about two to five peripheral lipid bilayers with a large, unstructured aqueous center. Lipid droplets, e.g., oil droplets, may be suspended in the aqueous center, leading to very high uptake of aqueous or lipophilic materials. Paucilamellar vesicles range from about 2-15.mu. in diameter.
Each type of lipid vesicle has distinct advantages for certain uses. Because of the relatively large amount of lipid in the lipid bilayers of the MLV's, they are considered best for encapsulation or transportation of lipophilic materials. The LUV's, because of their large aqueous/lipid volume ratio, are considered best for encapsulation of hydrophilic molecules, particularly macromolecules. SUV's have the advantage of small size which allows relatively easy access to the cells of tissue, but their small volume limits delivery of hydrophilic aqueous materials to trace amounts. However, SUV's may be useful in place of MLV's for the transportation of small quantities of lipophilic materials because of high lipid/water ratios. PLV's can transport large quantities of aqueous or lipophilic materials but their large size can preclude approach to certain tissues.
The present invention pertains to the formation of MLV's and PLV's. Since SUV's are commonly made by sonification of multilamellar lipid vesicles, it follows that the processes by which MLV's are produced can be used as part of a technique for making SUV's.
The conventional approach to producing multilamellar lipid vesicles, particularly liposomes made of phospholipids, starts by dissolving the lipids, together with any lipophilic additives, in an organic solvent. The organic solvent is then removed by evaporation using heat or by passing a stream of an inert gas (e.g., nitrogen) over the dissolved lipids. The residue is then hydrated with an aqueous phase, generally containing electrolytes and additives such as hydrophilic biologically-active materials, to form large multilamellar lipid membrane structures. In some variations, different types of particulate matter or structures have been used during the evaporation process to assist in the formation of the lipid residue. Those in the field have shown that by changing the physical structure of the lipid residue, better vesicles form upon hydration. Two recent review publications, Szoka and Papahdjopoulos, Ann. Rev. Biophys. Bioeng. 9:467-508 (1980), and Dousset and Douste-Blazy (in Les Liposomes, Puisieux and Delattre, Editors, techniques et Documentation Lavoisier, Paris, pp.41-73 (1985)), summarize the methods which have been used to make MLV's.
No matter how the MLV's or PLV's are formed, once made it is necessary to determine the effectiveness of the process. Two measurements commonly used to determine the effectiveness of encapsulation of materials in lipid vesicles are the encapsulated mass and captured volume. The encapsulated mass is the mass of the substance encapsulated per unit mass of the lipid and is often given as a percentage. The captured volume is defined as the amount of the aqueous phase trapped inside the vesicle divided by the amount of lipid in the vesicle structure, normally given in ml liquid/g lipid.
A disadvantage associated with producing MUV's or PLV's using standard methods is that these processes are costly, slow and relatively inefficient in terms of material. For example, the standard time to manufacture MLV's is in the order 2-20 hours. If SUV's are required, the sonification which breaks the multilamellar lipid structures into SUV's takes additional time. This slow processing is unwieldy and expensive for any large scale production of lipid vesicles.
While rapid, continuous-flow, mixing processes are known in other arts, e.g., chemical kinetics, the adaptation of such processes for the production of lipid vesicles has not heretofore been devised, or even suggested.
Accordingly, an object of the invention is to provide an improved method and apparatus for making multilamellar or paucilamellar lipid vesicles.
A further object of the invention is to provide a lipid vesicle forming technique which lends itself to commercial, high-volume production.
Another object of the invention to provide a method and apparatus for the rapid, efficient encapsulation of biologically-active macromolecules into lipid vesicles.
These and other objects and features of the invention will be apparent from the following description and drawings.